Proteomics-based investigation in C2C12 myoblast differentiation

Abstract

Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified.To further highlight this process, we studied cells at undifferentiated, intermediate and highly differentiated stages, and we analyzed, for each condition, morphological and proteomic changes. We also identified the proteins that showed statistical significant changes by a ESI-Q-TOF mass spectrometer. This work provides further evidence of the involvement of particular proteins in skeletal muscle development. Furthermore, the high level of expression of many heat shock proteins, suggests a relationship between differentiation and cellular stress. Intriguingly, the discovery of myogenesis-correlated proteins, known to play a role in apoptosis, suggests a link between differentiation and this type of cell death.

Keywords: 2-D electrophoresis; C2C12 cells; TEM; differentiation; mass spectrometry.; proteome.

Figure 1

C2C12 differentiation peculiarities, identified by TEM (A,B,C,E,F) and confocal microscope IF (D) at early (A), intermediate (B,C,D) and late (E,F) time. Cell membrane contacts (→) are followed by the fusion of focal membrane areas (*). α Sarcomeric actin can also be identified as stress fibers, well recognizable at TEM (➭), and as strongly fluorescent cytoplasmic spots at IF (D), at the intermediate stage. Late differentiation is characterized by the appearance of apoptotic cells (ap) and by the significantly increased number of mitochondria (m). A–F, bar = 1 µm

Figure 2

2-DE of 45 µg of C2C12 myoblast during differentiation. (A) Undifferentiated stage (time 0); (B) Early intermediate differentiation stage (3 days); (C) Late intermediate differentiation stage (5 days); (D) High differentiation phase (7 days). Gels were stained with silver nitrate.